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| Status |
Public on Jun 03, 2014 |
| Title |
OCILY3_BATF |
| Sample type |
SRA |
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| Source name |
ABC DLBCL
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| Organism |
Homo sapiens |
| Characteristics |
cell line: OCI-LY3
cell type: Diffuse large B-cell lymphoma (DLBCL)
chip antibody: sc-100974X, Santa Cruz
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| Treatment protocol |
SPIB knockdown on OCI-Ly3 were obtained by transfecting SPIB siRNA s13354 and scramble siRNA (Ambion, Life Technologies™) using Amaxa® Nucleofector® system and Amaxa® Cell line Nucleofector® Kit V (Lonza), setting D.023. Cells were harvested after 48hrs and the efficiency and the degree of the knockdown was assessed by RT-PCR and Western blot.
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| Growth protocol |
OCI-Ly3 and OCI-Ly10 were grown in IMDM with Glutamax™ (Life Technologies™), 10% HIFBS, 50µM betamercaptoethanol. Each batch of chromatin from each cell line were harvested from 2*10^7 cells. Batches were pooled together and distributed in aliquots. Individual CHIP were obtained from 4*10^6 cells.
H929 were grown in RPMI 1640, 10% HIFBS, 1% L-Glutamine. Each batch of chromatin were harvested from 2*10^7 cells. Batches were pooled together and distributed in aliquots. Individual CHIP were obtained from 4*10^6 cells.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell fixation and chromatin sonication were optimised for each cell line in order to obtain enrichment of DNA fragments between 200-500bp. Chromatin was pre-cleared with BSA saturated Protein A sepharose (Thermo Scientific). A fraction of the chromatin was then incubated overnight at 4°C with the appropriate antibody (in duplicate) and then immunoprecipitated with BSA saturated Protein A sepharose (Thermo Scientific) for 4 hours at 4°C. Following washing to remove aspecificity, each IP was eluted from the Protein A sepharose overnight. DNA was then purified with standard phenol/chloroform extraction and quantified using Quant-iT™ Picogreen® dsDNA Broad-Range Assay Kit (Life Technologies™).
IRF4, PU.1 and SPIB CHIP-seq libraries from OCI-LY3, OCI-LY10 and H929 were generated using the Illumina ChIP-seq Sample Prep Kit (Illumina®) according to manufacturer’s instructions. The BATF and the SPIB knockdown libraries were generated using the Microplex Library Preparation™ Kit (Diagenode) following manufacturer's instructions, and size selected using Agencourt® AMPure® XP beads (Beckman Coulter®).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
ASCII shift tag: 33
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| Data processing |
Basecalls performed using CASAVA version 1.6 and 1.8 (see ASCII shift tag of RAW files; 64 for version 1.6 of CASAVA and 33 for version 1.8)
If required reads were converted to fastq format (Solexa --> Sanger quality score)
Before aligning, reads were trimmed to remove adapters and low confidence regions using a python script. A 4 base sliding window was run along each read, if the average Q Phred score for a window was < 20 the read was trimmed at the window start, finally any match to adapter sequences were trimmed
Trimmed reads were aligned using Bowtie2 with the --very-senstive parameter. The resultant SAM files were converted to BAM using Samtools with the quality filter set to 20 (i.e. -q 20)
The BAM files were analysed for peaks using GEM, with quality filter set to 1 (i.e. -q 1)
ChIP-seq quality was assessed by cross-correlation using the MaSC and Phantompeakqual programs. Reads were extended to the length reported by MaSC, scaled to reads per million and converted to BigWig files. These were used for visualisation of the data and also for fold-change analysis for the SPIB knock-down ChIP-seq data.
Genome_build: hg19
Supplementary_files_format_and_content: BED files (converted from GEM events); BigWig coverage files (Used for ChIP-seq fold change analysis)
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| Submission date |
Apr 16, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Matthew Anthony Care |
| E-mail(s) |
matthew.care@york.ac.uk
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| Organization name |
The University of York
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| Department |
Department of Biology
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| Lab |
Bioscience Technology Facility, Data Science Hub
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| Street address |
Wentworth Way
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| City |
York |
| State/province |
North Yorkshire |
| ZIP/Postal code |
YO10 5DD |
| Country |
United Kingdom |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE56857 |
SPIB and BATF provide alternate determinants of IRF4 occupancy in Diffuse Large B-cell Lymphoma linked to disease heterogeneity |
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| Relations |
| BioSample |
SAMN02728902 |
| SRA |
SRX518668 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1370272_OCILY3_BATF_GEM_events.bed.gz |
91.2 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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